Protein Synthesis

Introduction

Protein Synthesis in
PROKARYOTES
Protein Biosynthesis Takes Place in Five Stages Activation of Amino Acids
Initiation
Elongation
Termination and Release
Folding and Posttranslational Processing

Activation of Amino Acids

Two fundamental chemical requirements must
be met:
(1) The carboxyl group of each amino acid must be
activated to facilitate formation of a peptide
bond
(2) A link must be established between each new
amino acid and the information in the mRNA
that encodes it.

 Both these requirements are met by attaching the amino

acid to a tRNA in the first stage of protein synthesis.
Reaction in cytosol
For each 20 amino acids attachment tRNA requires
APT and Mg2
Enzyme- aminoacyl tRNA synthetase

Initiation

AUG initiation codon specifies an amino - terminal methionine

residue methionine residue are of two types-

1) Methionine residue used in the middle of the mRNA seq (ie.,

apart from the inittaition site)

2) N-formylmethionyl-tRNAfMet (fMet-tRNAfMet)- used at

initiation site

 Two successive reactions.

First, methionine is attached to tRNA by the Met-tRNA
synthetase
A transformylase transfers a formyl group from N10formyltetrahydrofolate to the amino group of the Met residue

N-formyl group to the amino group of methionine by the

transformylase prevents fMet from entering interior positions in
a polypeptide

Initiation The initiation of polypeptide synthesis in bacteria requires The 30S ribosomal subunit
The mRNA coding for the polypeptide to be made
The initiating fMet-tRNAfMet
A set of three proteins called initiation factors (IF-1, IF-2, and IF-3),
GTP
The 50S ribosomal subunit,
Mg2.

 70s(sedimentation
coefficient) Ribosomes
splits to 30s and 50s
Ribosomes

 In step 1-

The 30S ribosomal subunit binds two initiation

factors
IF-1 and IF-3.
Factor IF-3 prevents the 30S and 50S subunits from
combining prematurely.
The mRNA then binds to the 30S subunit.

 The initiating (5)AUG is guided to its correct position by the

Shine- Dalgarno sequence in the mRNA
This consensus sequence is an initiation signal of four to nine
purine residues, 8 to 13 bp to the 5 side of the initiation codon.
It base pairs with 16srRNA which is present in the 30s
Ribosomes.
This mRNA-rRNA interaction positions the initiating (5)AUG
sequence of the mRNA in the precise position on the 30S
subunit where it is required for initiation of translation

 Bacterial ribosomes have three sites that bind aminoacyltRNAsAminoacyl (A) site
peptidyl (P) site
Exit (E) site

Factor IF-1 binds at the A site and Prevents tRNA binding at

this site during initiation
The initiating (5)AUG is positioned at the P site, the only site
to which fMet tRNAcan bind

 The fMet-tRNAfMet is the only aminoacyl-tRNA that binds

first to the P site

During the subsequent elongation stage, all other incoming

aminoacyl-tRNAs (including the Met-tRNAMet that binds to
interior AUG codons) bind first to the A site and only
subsequently to the P and E sites.

The E site is the site from which the uncharged tRNAs leave
during elongation

 In step 2 The complex consisting of the 30S ribosomal subunit, IF-3,

and mRNA is joined by both GTP-bound IF-2 and the
initiating fMet-tRNAfMet. The anticodon of this tRNA now
pairs correctly with the mRNAs initiation codon.
In step 3
This large complex combines with the 50S ribosomal subunit;
simultaneously, the GTP bound to IF-2 is hydrolyzed to GDP
and Pi, which are released from the complex. All three
initiation factors depart from the ribosome at this point.

 Completion of the steps in produces a functional 70S ribosome

called the initiation complex, containing the mRNA and the
initiating fMettRNAfMet

Elongation
Elongation requires The initiation complex described above
Aminoacyl-tRNA
Set of three soluble cytosolic proteins called elongation
factors (EF-Tu, EF-Ts, and EF-G in bacteria)
GTP

 Elongation Step 1: Binding of an Incoming

Aminoacyl-tRNA
The EF-TuGTP complex is regenerated in a process
involving EF-Ts and GTP
The appropriate incoming aminoacyl-tRNA binds to a
complex of GTP-bound EF-Tu.
The resulting aminoacyl tRNA EF-TuGTP complex binds to
the A site of the 70S initiation complex.
The GTP is hydrolyzed and an EF-TuGDP complex is
released from the 70S ribosome

 Elongation Step 2: Peptide Bond Formation A peptide bond is now formed between the two amino acids
bound by their tRNAs to the A and P sites on the ribosome
The -amino group of the amino acid in the A site acts as a
nucleophile, displacing the tRNA in the P site to form the
peptide bond.
The enzymatic activity that catalyzes peptide bond formation
has historically been referred to as peptidyl transferase

 Elongation Step 3: Translocation

In the final step of the elongation cycle, translocation, the
ribosome moves one codon toward the 3 end of the mRNA
This movement shifts the anticodon of the dipeptidyltRNA,
which is still attached to the second codon of the mRNA, from
the A site to the P site, and shifts the deacylated tRNA from the
P site to the E site
From where the tRNA is released into the cytosol.

 The third codon of the mRNA now lies in the A site and the
second codon in the P site.

Movement of the ribosome along the mRNA requires EF-G

(also known as translocase) and the energy provided by
hydrolysis of another molecule of GTP.

Stage 4: Termination and Release

Three termination codons in the mRNA (UAA, UAG, UGA) (any

one)
In bacteria, once a termination codon occupies the ribosomal A site,

three termination factors, or release factorsthe proteins RF-1,

RF-2, and RF-3
(1) hydrolysis of the terminal peptidyltRNA bond
(2) release of the free polypeptide and the last tRNA, now

uncharged, from the P site;

(3) dissociation of the 70S ribosome into its 30S and 50S subunits,

ready to start a new cycle of polypeptide synthesis

 RF-1 recognizes the termination codons UAG and

UAA, and RF-2 recognizes UGA and UAA.
Either RF-1 or RF-2 (depending on which codon is
present) binds at a termination codon and induces
peptidyl transferase to transfer the growing
polypeptide to a water molecule rather than to
another amino acid.

Protein Synthesis in
EUKARYOTES

Initiation
80S ribosomal splits to 60S and 40S ribosomal units

Eukaryotic mRNAs are bound to the ribosome as a complex

with a number of specific binding proteins.

Several of these tie together the 5 and 3 ends of the message. At

the 3 end, the mRNA is bound by the poly(A) binding protein
(PAB).

 Eukaryotic cells have at least nine initiation factors

A complex called eIF4F
proteins eIF4E, eIF4G, and eIF4A,
eIF4E binds to the 5 cap
eIF4G binds to both eIF4E and PAB,
effectively tying them together.
eIF4A has an RNA helicase activity.

eIF4F associates with another factor, eIF3, and with the 40S
ribosomal subunit

 The initiating (5)AUG is detected within the mRNA not

by its proximity to a Shine-Dalgarno-like sequence but by
a scanning process:
eIF4F complex is involved in this process

Elongation

The elongation cycle in eukaryotes is quite similar to that in prokaryotes.

Three eukaryotic elongation factors (eEF1, eEF1, and eEF2) have

functions analogous to those of the bacterial elongation factors (EF-Tu,
EF-Ts, and EF-G, respectively).

Eukaryotic ribosomes do not have an E site; uncharged tRNAs are

expelled directly from the P site

Termination
In eukaryotes, a single release factor, eRF, recognizes all
three termination codons.

Folding and Posttranslational

Processing

STEPS

1. Amino-Terminal and Carboxyl-Terminal Modifications

2. Loss of Signal Sequences
3. Modification of Individual Amino Acids4. Attachment of Carbohydrate Side Chains5. Addition of Isoprenyl Groups6. Addition of Prosthetic Groups
7. Proteolytic Processing
8. Formation of Disulfide Cross Links

 Amino-Terminal and Carboxyl-Terminal

Modifications

The formyl group, the amino-terminal Met residue,

and often additional amino-terminal (and, in some

cases, carboxyl-terminal) residues
Removed enzymatically in formation of the final
functional protein

 Loss of Signal Sequences

15 to 30 residues at the amino-terminal end of some
proteins play a role in directing the protein to its ultimate
destination in the cell.

These residues ultimately removed by specific peptidases

 Modification of Individual Amino Acids Ser,

Thr,

and

Tyr

residues

are

enzymatically

phosphorylated by ATP-

Example, the milk protein casein has many phosphoserine

groups that bind Ca2. Calcium, phosphate, and amino acids are
all valuable to suckling young, so casein efficiently provides
three essential nutrients.

 Attachment of Carbohydrate Side Chains The carbohydrate side chains of glycoproteins are
attached covalently during or after synthesis of the
polypeptide.
Many proteins that function extracellularly, as well as the
lubricating proteoglycans that coat mucous membranes, contain
oligosaccharide side chains

 Addition of Isoprenyl Groups A number of eukaryotic proteins are modified by the

addition of groups derived from isoprene (isoprenyl
groups: helps to anchor the protein in a membrane).
Proteins modified in this way include the Ras proteins, products
of the ras oncogenes and proto-oncogenes, and G proteins.

 Addition of Prosthetic Groups

Many prokaryotic and eukaryotic proteins require for
their activity covalently bound prosthetic groups.

Two examples are the biotin molecule of acetyl-CoA carboxylase

and the heme group of hemoglobin or cytochrome c.

Proteolytic Processing
Many proteins are initially synthesized as large, inactive
precursor polypeptides that are proteolytically trimmed to
form their smaller, active forms.

Examples include proinsulin

Formation of Disulfide Cross Links

After folding into their native conformations, some
proteins form intrachain or interchain disulfide bridges
between Cys residues

The cross-links formed in this way help to protect the

native conformation of the protein molecule from
denaturation in the extracellular environment

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